scholarly journals Editing of the TRIM5 Gene Decreases the Permissiveness of Human T Lymphocytic Cells to HIV-1

Viruses ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 24
Author(s):  
Kevin Désaulniers ◽  
Levine Ortiz ◽  
Caroline Dufour ◽  
Alix Claudel ◽  
Mélodie B. Plourde ◽  
...  
Keyword(s):  
Type I Interferons ◽  
Type I ◽  
Gene Products ◽  
Binding Region ◽  
Homology Directed Repair ◽  
Effector Genes ◽  
Cell Clones ◽  
Innate Effector ◽  
Isolated Cell ◽  
Hiv 1

Tripartite-motif-containing protein 5 isoform α (TRIM5α) is a cytoplasmic antiretroviral effector upregulated by type I interferons (IFN-I). We previously showed that two points mutations, R332G/R335G, in the retroviral capsid-binding region confer human TRIM5α the capacity to target and strongly restrict HIV-1 upon overexpression of the mutated protein. Here, we used clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated homology-directed repair (HDR) to introduce these two mutations in the endogenous human TRIM5 gene. We found 6 out of 47 isolated cell clones containing at least one HDR-edited allele. One clone (clone 6) had both alleles containing R332G, but only one of the two alleles containing R335G. Upon challenge with an HIV-1 vector, clone 6 was significantly less permissive compared to unmodified cells, whereas the cell clones with monoallelic modifications were only slightly less permissive. Following interferon (IFN)-β treatment, inhibition of HIV-1 infection in clone 6 was significantly enhanced (~40-fold inhibition). TRIM5α knockdown confirmed that HIV-1 was inhibited by the edited TRIM5 gene products. Quantification of HIV-1 reverse transcription products showed that inhibition occurred through the expected mechanism. In conclusion, we demonstrate the feasibility of potently inhibiting a viral infection through the editing of innate effector genes. Our results also emphasize the importance of biallelic modification in order to reach significant levels of inhibition by TRIM5α.

scholarly journals Editing of the TRIM5 gene decreases the permissiveness of human T lymphocytic cells to HIV-1

2020 ◽  
Author(s):  
Kevin Désaulniers ◽  
Levine Ortiz ◽  
Caroline Dufour ◽  
Alix Claudel ◽  
Mélodie B. Plourde ◽  
...  
Keyword(s):  
Type I Interferons ◽  
Type I ◽  
Gene Products ◽  
Over Expression ◽  
Homology Directed Repair ◽  
Effector Genes ◽  
Cell Clones ◽  
Innate Effector ◽  
Isolated Cell ◽  
Hiv 1

AbstractTRIM5α is a cytoplasmic antiretroviral effector upregulated by type I interferons (IFN-I). We previously showed that two points mutations, R332G/R335G, in the retroviral capsid-binding region confer human TRIM5α the capacity to target and strongly restrict HIV-1 upon over-expression of the mutated protein. Here, we used CRISPR-Cas9-mediated homology-directed repair (HDR) to introduce these two mutations in the endogenous human TRIM5 gene. We found 6 out of 47 isolated cell clones containing at least one HDR-edited allele. One clone (Clone 6) had both alleles containing R332G but only one of the two alleles containing R335G. Upon challenge with an HIV-1 vector, clone 6 was significantly less permissive compared to unmodified cells, whereas the cell clones with monoallelic modifications were only slightly less permissive. Following IFN-β treatment, inhibition of HIV-1 infection in clone 6 was significantly enhanced (~40-fold inhibition). TRIM5α knockdown confirmed that HIV-1 was inhibited by the edited TRIM5 gene products. Quantification of HIV-1 reverse transcription products showed that inhibition occurred through the expected mechanism. In conclusion, we demonstrate the feasibility of potently inhibiting a viral infection through editing of innate effector genes. Our results also emphasize the importance of biallelic modification in order to reach significant levels of inhibition by TRIM5α.

scholarly journals Editing of the Human TRIM5 Gene Decreases the Permissiveness of Jurkat T Lymphocytic Cells to HIV-1

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 96
Author(s):  
Kevin Désaulniers ◽  
Levine Ortiz ◽  
Caroline Dufour ◽  
Alix Claudel ◽  
Mélodie B. Plourde ◽  
...  
Keyword(s):  
Point Mutations ◽  
Type I Interferons ◽  
Type I ◽  
Retroviral Transduction ◽  
Specific Pcr ◽  
Effector Genes ◽  
Cell Clones ◽  
Core Components ◽  
Innate Effector ◽  
Hiv 1

TRIM5α is a cytoplasmic antiviral effector induced by type I interferons (IFN-I) that has the potential to intercept incoming retroviruses by interacting with their capsid core, leading to uncoating induction and the partial degradation of core components. Most HIV-1 strains escape restriction by human TRIM5α due to a lack of interaction between TRIM5α and its viral molecular target. We previously showed, however, that two point mutations, R332G/R335G, in the capsid-binding region confer human TRIM5α with the capacity to target and strongly restrict HIV-1 upon the overexpression of the mutated protein. Here, we explored the possibility to introduce these two mutations in the endogenous human TRIM5 gene by CRISPR-Cas9-mediated gene editing. For this, we electroporated CRISPR ribonucleoproteins (RNPs) and the donor DNA into Jurkat T lymphocytic cells and isolated clones by limiting dilution. We analyzed 47 clones using specific PCR assays, and found that six clones (13%) contained at least one gene-edited allele. One clone (clone 6) had both alleles edited for R332G, but only one of the two alleles was edited for R335G. Upon challenge with an HIV-1 vector, clone 6 was significantly less permissive compared to unmodified cells, whereas the cell clones with monoallelic modifications were only slightly less permissive. Following IFN-β treatment, the inhibition of HIV-1 infection in clone 6 was significantly enhanced (~50-fold inhibition), whereas IFN-β treatment had no effect on TRIM5α overexpressed by retroviral transduction. Knockdown experiments confirmed that HIV-1 was inhibited by the edited TRIM5 gene products, whereas quantification of HIV-1 reverse transcription products confirmed that inhibition occurred through the expected mechanism. In conclusion, we demonstrate the feasibility of potently inhibiting a viral infection through the editing of innate effector genes, but our results also emphasize the importance of biallelic modification in order to reach significant levels of inhibition by TRIM5α.

scholarly journals Editing of the Human TRIM5 Gene to Introduce Mutations with the Potential to Inhibit HIV-1

2017 ◽  
Author(s):  
Caroline Dufour ◽  
Alix Claudel ◽  
Nicolas Joubarne ◽  
Natacha Merindol ◽  
Tara Maisonnet ◽  
...  
Keyword(s):  
Cell Line ◽  
Type I Interferon ◽  
Human Cells ◽  
Restriction Factor ◽  
Type I ◽  
Dna Transfection ◽  
Homology Directed Repair ◽  
Cell Clones ◽  
Overall Efficiency ◽  
Hiv 1

ABSTRACTThe type I interferon (IFN-I)-inducible human restriction factor TRIM5α inhibits the infection of human cells by specific nonhuman retroviruses, such as N-MLV and EIAV, but does not generally target HIV-1. However, the introduction of two aminoacid substitutions, R332G and R355G, in the human TRIM5α (huTRIM5α) domain responsible for retroviral capsid recognition leads to efficient HIV-1 restriction. Using a DNA transfection-based CRISPR-Cas9 genome editing protocol, we successfully mutated TRIM5 to its HIV-1-restrictive version by homology-directed repair (HDR) in HEK293T cells. Nine clones bearing at least one HDR-edited TRIM5 allele containing both mutations were isolated (5.6% overall efficiency), whereas another one contained only the R332G mutation. Of concern, several of these HDR-edited clones contained on-target undesired mutations, and none had all the alleles corrected. We observed a lack of HIV-1 restriction in the cell clones generated, even when cells were stimulated with IFN-I prior to infection. This, however, was partly explained by the unexpectedly low potential for TRIM5α-mediated restriction activity in this cell line. Our study demonstrates the feasibility of editing the TRIM5 gene to in human cells and identifies the main challenges to be addressed in order to use this approach to confer protection from HIV-1.

scholarly journals HIV-Infected Macrophages Are Infected and Killed by the Interferon-Sensitive Rhabdovirus MG1

2021 ◽  
Vol 95 (9) ◽  
Author(s):  
Teslin S. Sandstrom ◽  
Nischal Ranganath ◽  
Stephanie C. Burke Schinkel ◽  
Syim Salahuddin ◽  
Oussama Meziane ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Oncolytic Viruses ◽  
Type I Interferons ◽  
Viral Reservoir ◽  
Type I ◽  
Antiviral Signaling ◽  
Infected Cells ◽  
Hiv 1

ABSTRACT The use of unique cell surface markers to target and eradicate HIV-infected cells has been a longstanding objective of HIV-1 cure research. This approach, however, overlooks the possibility that intracellular changes present within HIV-infected cells may serve as valuable therapeutic targets. For example, the identification of dysregulated antiviral signaling in cancer has led to the characterization of oncolytic viruses capable of preferentially killing cancer cells. Since impairment of cellular antiviral machinery has been proposed as a mechanism by which HIV-1 evades immune clearance, we hypothesized that HIV-infected macrophages (an important viral reservoir in vivo) would be preferentially killed by the interferon-sensitive oncolytic Maraba virus MG1. We first showed that HIV-infected monocyte-derived macrophages (MDM) were more susceptible to MG1 infection and killing than HIV-uninfected cells. As MG1 is highly sensitive to type I interferons (IFN-I), we then investigated whether we could identify IFN-I signaling differences between HIV-infected and uninfected MDM and found evidence of impaired IFN-α responsiveness within HIV-infected cells. Finally, to assess whether MG1 could target a relevant, primary cell reservoir of HIV-1, we investigated its effects in alveolar macrophages (AM) obtained from effectively treated individuals living with HIV-1. As observed with in vitro-infected MDM, we found that HIV-infected AM were preferentially eliminated by MG1. In summary, the oncolytic rhabdovirus MG1 appears to preferentially target and kill HIV-infected cells via impairment of antiviral signaling pathways and may therefore provide a novel approach to an HIV-1 cure. IMPORTANCE Human immunodeficiency virus type 1 (HIV-1) remains a treatable, but incurable, viral infection. The establishment of viral reservoirs containing latently infected cells remains the main obstacle in the search for a cure. Cure research has also focused on only one cellular target of HIV-1 (the CD4+ T cell) while largely overlooking others (such as macrophages) that contribute to HIV-1 persistence. In this study, we address these challenges by describing a potential strategy for the eradication of HIV-infected macrophages. Specifically, we show that an engineered rhabdovirus—initially developed as a cancer therapy—is capable of preferential infection and killing of HIV-infected macrophages, possibly via the same altered antiviral signaling seen in cancer cells. As this rhabdovirus is currently being explored in phase I/II clinical trials, there is potential for this approach to be readily adapted for use within the HIV-1 cure field.

scholarly journals Concurrent administration of IFNα14 and cART in TKO-BLT mice enhances suppression of HIV-1 viremia but does not eliminate the latent reservoir

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Kathrin Sutter ◽  
Kerry J. Lavender ◽  
Ronald J. Messer ◽  
Marek Widera ◽  
Katie Williams ◽  
...  
Keyword(s):  
Infection Type ◽  
Treatment Interruption ◽  
Type I Interferons ◽  
Latent Reservoir ◽  
Type I ◽  
Concurrent Administration ◽  
Viral Dna ◽  
Therapy Interruption ◽  
Hiv 1

AbstractCombination antiretroviral therapy (cART) prevents HIV-1 replication but does not eliminate the latent reservoir and cure the infection. Type I interferons (IFN) mediate antiviral effects through different mechanisms than cART. We previously showed that IFNα14 is the most potent IFNα subtype against HIV-1 and that it can significantly reduce the HIV-1 proviral reservoir. This study sought to determine whether combining cART with IFNα14 therapy would produce greater reductions in HIV-1 viral and proviral loads than ART alone. Immunodeficient Rag2−/−γc−/−CD47−/− C57BL/6 mice were humanized by the BLT method, infected with HIV-1JR-CSF and the in vivo efficacy of cART was compared with combined cART/IFNα14 therapy. Infection was allowed to establish for 6 weeks prior to 4 weeks of treatment with oral cART either with or without IFNα14. Plasma viral RNA and splenic CD4+ T cell viral DNA levels were measured immediately after treatment and after 2 weeks of therapy interruption. Augmentation of cART with IFNα14 resulted in significantly enhanced suppression of HIV-1 plasma viremia. However, no significant reduction in total viral DNA was detectable. Furthermore, virus rebounded after treatment interruption to similar levels in both groups. Thus, augmentation of cART with IFNα14 resulted in a more pronounced reduction of HIV viremia levels over cART alone, but the effect was not potent enough to be detected at the viral DNA level or to prevent virus rebound following therapy interruption in immune system-humanized mice.

Inhibition of Angiogenesis by Type I Interferons in Models of Kaposi'S Sarcoma

1999 ◽  
Vol 14 (4) ◽  
pp. 257-262 ◽  
Author(s):  
C. Marchisone ◽  
R. Benelli ◽  
A. Albini ◽  
L. Santi ◽  
D. M. Noonan
Keyword(s):  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Inhibitory Effect ◽  
Type I Interferons ◽  
Endothelial Cell Migration ◽  
Type I ◽  
Type I Ifns ◽  
Hiv 1

Kaposi's Sarcoma (KS) is a pathology which occurs with increased frequency and in a particularly aggressive form in AIDS patients. The HIV-1 Tat protein appears to be an important co-factor in the induction of the extensive neo-vascularization associated with AIDS-KS. Tat acts as a chemoattractant for endothelial cells in vitro, inducing both chemotactic and invasive responses. Several clinical trials have been performed testing the effectiveness of diverse biological agents in therapy of KS, among these the type I interferons. Type I IFNs have diverse biological functions besides their anti-viral activity, including anti-angiogenic properties. We have shown that IFNα and IFNβ are potent inhibitors of both primary and immortalized endothelial cell migration and morphogenesis in vitro as well as neo-angiogenesis induced by HIV-1 Tat in vivo. The inhibitory effect of IFN class I on HIV-Tat associated angiogenesis further supports its use as a therapy for epidemic Kaposi's sarcoma. The use of recombinant IFNs at the levels required to obtain a therapeutic effect are associated with side effects and toxicity, therefore we are now developing a gene therapy approach for constant and local delivery type I IFNs.

scholarly journals Activation of a dendritic cell–T cell axis by Ad5 immune complexes creates an improved environment for replication of HIV in T cells

2008 ◽  
Vol 205 (12) ◽  
pp. 2717-2725 ◽  
Author(s):  
Matthieu Perreau ◽  
Giuseppe Pantaleo ◽  
Eric J. Kremer
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Complexes ◽  
Vaccine Trial ◽  
Type I Interferons ◽  
Type I ◽  
Cell Axis ◽  
Vector Vaccine ◽  
Hiv 1

The STEP HIV vaccine trial, which evaluated a replication-defective adenovirus type 5 (Ad5) vector vaccine, was recently stopped. The reasons for this included lack of efficacy of the vaccine and a twofold increase in the incidence of HIV acquisition among vaccinated recipients with increased Ad5-neutralizing antibody titers compared with placebo recipients. To model the events that might be occurring in vivo, the effect on dendritic cells (DCs) of Ad5 vector alone or treated with neutralizing antiserum (Ad5 immune complexes [IC]) was compared. Ad5 IC induced more notable DC maturation, as indicated by increased CD86 expression, decreased endocytosis, and production of tumor necrosis factor and type I interferons. We found that DC stimulation by Ad5 IC was mediated by the Fcγ receptor IIa and Toll-like receptor 9 interactions. DCs treated with Ad5 IC also induced significantly higher stimulation of Ad5-specific CD8 T cells equipped with cytolytic machinery. In contrast to Ad5 vectors alone, Ad5 IC caused significantly enhanced HIV infection in DC–T cell cocultures. The present results indicate that Ad5 IC activates a DC–T cell axis that, together with the possible persistence of the Ad5 vaccine in seropositive individuals, may set up a permissive environment for HIV-1 infection, which could account for the increased acquisition of HIV-1 infection among Ad5 seropositive vaccine recipients.

scholarly journals Type I interferons suppress viral replication but contribute to T cell depletion and dysfunction during chronic HIV-1 infection

JCI Insight ◽  
2017 ◽  
Vol 2 (12) ◽  
Author(s):  
Liang Cheng ◽  
Haisheng Yu ◽  
Guangming Li ◽  
Feng Li ◽  
Jianping Ma ◽  
...  
Keyword(s):  
T Cell ◽  
Viral Replication ◽  
Type I Interferons ◽  
Cell Depletion ◽  
Type I ◽  
T Cell Depletion ◽  
Hiv 1

scholarly journals Does the Cytoplasmic Tail Matter? Mechanism of Viral Envelope Glycoprotein Targeting by Membrane-Associated-RING-CH (MARCH) Proteins

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 21
Author(s):  
Cheng man Lun ◽  
Abdul A. Waheed ◽  
Eric O. Freed
Keyword(s):  
Antiviral Activity ◽  
Envelope Glycoprotein ◽  
Protein Targeting ◽  
Viral Envelope ◽  
Surface Proteins ◽  
Type I Interferons ◽  
Type I ◽  
Endogenous Expression ◽  
Viral Glycoproteins ◽  
Hiv 1

The MARCH family of RING-finger E3 ubiquitin ligases comprise 11 members that have been reported to play a variety of roles in the downregulation of cell-surface proteins involved in adaptive immunity. The RING-CH domain of MARCH proteins is thought to ubiquitinate the cytoplasmic tails (CTs) of target proteins, leading to protein degradation through either lysosomal or proteasomal pathways. Three MARCH proteins (MARCH1, 2, and 8) have recently been reported to target the HIV-1 envelope glycoprotein (Env) and vesicular stomatitis virus G glycoprotein (VSV-G), thereby impairing the infectivity of HIV-1 virions bearing HIV-1 Env or VSV-G. However, the mechanism of antiviral activity remains poorly defined. Our data show that MARCH proteins antagonize the full-length forms of HIV-1 Env, VSV-G, and Ebola glycoprotein (GP), and impair the infectivity of HIV-1 virions bearing these viral glycoproteins. This Env-targeting activity of the MARCH proteins requires the E3 ubiquitin ligase activity of the RING-CH domain. We observe that the MARCH protein targeting of VSV-G is, to a large extent, CT-dependent. In striking contrast, the MARCH-protein targeting of HIV-1 Env and Ebola GP does not require the CT. Confocal microscopy data demonstrate that MARCH proteins are able to trap the viral glycoproteins in an intracellular compartment. We observe that the endogenous expression of MARCH8 in T-cell lines and PBMCs is inducible by type I interferons (a and b) and is also upregulated by HIV-1 infection. Current studies are aimed at identifying the cellular target for MARCH-mediated ubiquitination in the context of their antiviral activity. These results will clarify the mechanism by which MARCH proteins antagonize viral glycoproteins and provide insights into the antiviral role of cellular inhibitory factors in Env biogenesis, trafficking, and virion incorporation.

scholarly journals Host and Viral Determinants of Mx2 Antiretroviral Activity

2014 ◽  
Vol 88 (14) ◽  
pp. 7738-7752 ◽  
Author(s):  
Idoia Busnadiego ◽  
Melissa Kane ◽  
Suzannah J. Rihn ◽  
Hannah F. Preugschas ◽  
Joseph Hughes ◽  
...  
Keyword(s):  
Therapeutic Potential ◽  
Early Stage ◽  
Type I Interferons ◽  
Primate Species ◽  
Type I ◽  
Viral Determinants ◽  
Viral Target ◽  
Hiv 1 ◽  
Antiretroviral Activity

ABSTRACTMyxovirus resistance 2 (Mx2/MxB) has recently been uncovered as an effector of the anti-HIV-1 activity of type I interferons (IFNs) that inhibits HIV-1 at an early stage postinfection, after reverse transcription but prior to proviral integration into host DNA. The mechanistic details of Mx2 antiviral activity are not yet understood, but a few substitutions in the HIV-1 capsid have been shown to confer resistance to Mx2. Through a combination ofin vitroevolution and unbiased mutagenesis, we further map the determinants of sensitivity to Mx2 and reveal that multiple capsid (CA) surfaces define sensitivity to Mx2. Intriguingly, we reveal an unanticipated sensitivity determinant within the C-terminal domain of capsid. We also report that Mx2s derived from multiple primate species share the capacity to potently inhibit HIV-1, whereas selected nonprimate orthologs have no such activity. Like TRIM5α, another CA targeting antiretroviral protein, primate Mx2s exhibit species-dependent variation in antiviral specificity against at least one extant virus and multiple HIV-1 capsid mutants. Using a combination of chimeric Mx2 proteins and evolution-guided approaches, we reveal that a single residue close to the N terminus that has evolved under positive selection can determine antiviral specificity. Thus, the variable N-terminal region can define the spectrum of viruses inhibited by Mx2.IMPORTANCEType I interferons (IFNs) inhibit the replication of most mammalian viruses. IFN stimulation upregulates hundreds of different IFN-stimulated genes (ISGs), but it is often unclear which ISGs are responsible for inhibition of a given virus. Recently, Mx2 was identified as an ISG that contributes to the inhibition of HIV-1 replication by type I IFN. Thus, Mx2 might inhibit HIV-1 replication in patients, and this inhibitory action might have therapeutic potential. The mechanistic details of how Mx2 inhibits HIV-1 are currently unclear, but the HIV-1 capsid protein is the likely viral target. Here, we determine the regions of capsid that specify sensitivity to Mx2. We demonstrate that Mx2 from multiple primates can inhibit HIV-1, whereas Mx2 from other mammals (dogs and sheep) cannot. We also show that primate variants of Mx2 differ in the spectrum of lentiviruses they inhibit and that a single residue in Mx2 can determine this antiviral specificity.

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